Wnt-induced Vangl2 phosphorylation is dose-dependently required for planar cell polarity in mammalian development

Abstract

Planar cell polarity (PCP) is an evolutionarily conserved essential mechanism that provides directional information to control and coordinate polarized cellular and tissue behavior during embryonic development. Disruption of PCP leads to severe morphological defects in vertebrates and its dysregulation results in a variety of human diseases such as neural tube defects and skeletal dysplasia. PCP is governed by a set of highly conserved core proteins that are asymmetrically localized at the cell surface throughout the polarized tissues. The uniform directionality of PCP is established by global cues, such as Wg/Wnt signaling gradients that break the original symmetrical localization of core PCP proteins including Vang/Vangl and Fz/Fzd. However, the exact mechanism remains elusive. In this study, we found that Vangl2 phosphorylation, which was previously identified to be induced by Wnt5a signaling, is required for Vangl2 functions in mammalian PCP in multiple tissues. The in vivo activities of Vangl2 are determined by its phosphorylation level. Phospho-mutant Vangl2 exhibits dominant negative effects, whereas Vangl2 with reduced phosphorylation is hypomorphic. We show that Vangl2 phosphorylation is essential for its uniform polarization pattern. Moreover, serine/threonine kinases CK1ɛ and CK1δ are redundantly required for Wnt5a-induced Vangl2 phosphorylation. Dvl family members are also required for Wnt5a-induced Vangl2 phosphorylation by enhancing the interaction of CK1 and Vangl2. These findings demonstrate that induction of Vangl protein phosphorylation plays an essential role in transducing Wnt5a signaling to establish PCP in mammalian development, suggesting a phosphorylation-regulated "Vangl activity gradient" model in addition to the well-documented "Fz activity gradient" model in Wnt/PCP signaling.

Coordinated directional outgrowth and pattern formation by integration of Wnt5a and Fgf signaling in planar cell polarity

“…We then investigated the molecular mechanism by which PCP is regulated by Fgf signaling. As we have shown previously that Wnt5a induces Vangl2 phosphorylation, and we and others found that Vangl2 activities depend on its levels of phosphorylation in multiple organisms, including Drosophila, zebrafish, Xenopus and mouse (Gao et al, 2011;Ossipova et al, 2015;Kelly et al, 2016;Yang et al, 2017), we tested whether Fgf could also regulate Vangl2 phosphorylation in limb bud mesenchymal cells. We isolated mouse E10.5-E11.5 limb bud mesenchymal cells and treated them with recombinant Fgf8 and/or Wnt5a proteins.…”

Frizzled-dependent Planar Cell Polarity without Wnt Ligands

“…In fish and frog embryos, mutation or knock down of Wnt5a or Wnt11 lead to a reduction in the PCP-dependent cell movements that lead to axis elongation (Rauch et al, 1997;Heisenberg et al, 2000;Tada and Smith, 2000;Wallingford, Vogeli and Harland, 2001;Andre et al, 2015). Likewise, in the mouse limb bud, deletion of Wnt5a interferes with the establishment of PCP in chondrocytes along the proximal-distal axis and thus with tissue elongation (Gao et al, 2011;Yang et al, 2017). Therefore, in these instances, a Wnt ligand is required for PCP, although it is not clear whether this role is instructive or permissive (Gao, 2012;Lawrence and Casal, 2013).…”

Non-canonical Wnt signalling initiates scarring in biliary disease

“…Following Cre expression, VANGL2 protein and Vangl2 mRNA expression are lost (denoted as Vangl2 ΔTM , figure 5b and 5c), whilst Vangl1 mRNA remains unchanged (figure 5c). Vangl2 functions upstream of Jnk/cJUN signalling in embryonic development (18) and in breast cancer cells (26). We have shown that Porcupine--i alters Jnk/cJun in biliary epithelial cells, therefore we sought to determine whether this phenotype was mediated through Vangl2.…”

“…These so called non--canonical Wnt signalling pathways are highly conserved from Drosophila (13) through to vertebrates (14) and utilise Wnt ligands in combination with a broad range of cell surface receptors, to activate diverse downstream processes (15). The most widely reported non--canonical Wnt signalling cascades are planar cell polarity (PCP), which regulates activation of cJUN dependant transcription and cytoskeletal rearrangement through Rho and Rock (16)(17)(18), and Wnt/Ca 2+ signalling, which, though modulation of the cellular Ca 2+ concentration, activates NFAT dependant transcription (19)(20)(21). Other non--canonical Wnt signalling pathways have also been identified; for example activation of YAP/TAZ signalling though Rho dependant inhibition of Lats1/2, however it remains unclear how broadly applicable these pathways are (22).…”

“…These data suggest that Wnt5a represents an early response in biliary fibrosis that can be compensated for by other ligands later in disease or Wnt5a produced elsewhere, however, if these ligands act through common pathways then targeting non--canonical Wnt receptors would demonstrate a sustained anti--fibrotic response.Non--canonical Wnt receptors are expressed by biliary cells:The receptor complexes which mediate non--canonical Wnt signalling are diverse and it is unclear how they interact(15). However, a number of receptors, including Frizzled receptors and orphan receptor tyrosine kinases such as Ror1/2 and Ptk7, which themselves are known to bind Wnt5a, converge on the scaffolding protein VANGL2(18,49,50),(49) (schematic, figure 4a).In uninjured, 5 or 14 day DDC treated mice, expression of Wnt5a positively correlates with that of Vangl2 (figure 4b). Moreover, in patients with PSC, VANGL2 mRNA levels, but not those of the homologue VANGL1, were significantly increased when compared to livers without underlying biliary disease (supplementary figure 6a).…”